The mode and effect of antiviral nucleotide analogs, by AZT, ddI, 3TC, D4T and others on the inhibition and fidelity of the mitochondrial DNA polymerase and mitochondrial DNA replication have been documented and characterized in my laboratory. We now know what structural properties set this polymerase apart from the nuclear DNA polymerases to give rise to mitochondrial toxicity. We previously compared the inhibition, insertion, and exonucleolytic removal of five currently approved antiviral nucleotide analogs on the purified human recombinant DNA polymerase gamma. The apparent Km and kcat values were determined for the incorporation of TTP, dCTP, dGTP, 2-3-dideoxy-TTP (ddTTP), 3-azido-TTP (AZT-TP), 2-3-dideoxy-CTP (ddCTP), 2-3didehydro-TTP (D4T-TP), (-)-2,3-dideoxy-3-thiacytidine (3TC-TP), and carbocyclic 2,3-didehydro-dGTP (CBV-TP). Kinetic studies indicate that the apparent in vitro hierarchy of mitochondrial toxicity for the approved NRTIs is: ddC(zalcitabine) = ddI(didanosine) = D4T(stavudine) > >3TC(lamivudine) >PMPA(tenofovir)> AZT(zidovudine) > CBV(abacavir). The human pol gamma utilized dideoxynucleotides and D4T-TP in vitro as efficiently as the natural deoxynucleoside triphosphates, whereas AZT-TP, 3TC-TP and CBV-TP were moderate inhibitors of chain elongation. We have also identified genetic variants of the mitochondrial DNA polymerase that increases the susceptibility of these NRTI to cause mitochondrial toxicity and identified critical amino acids in the mitochondrial DNA polymerase that allow for insertion of these NRTIs into mitochondrial DNA. In collaboration with Miriam Poirier at the NCI, will are evaluating mitochondrial DNA for mutations and deletions from patas monkeys that have been exposed in utero to NRTIs. Pregnant patas monkeys were exposed with human equivalent doses of AZT, 3TC, abacavir and nevirapine. Tissues were collected at birth, 1 and 3 years of age and will be analyzed by next generation sequencing for point mutations and deletions in mitochondrial DNA. This analysis will help us to understand the long term consequences of NRTI treatment on children exposed in utero to antiretroviral therapy. We have also reviewed the evidence for additional DNA polymerases that may be imported into the mitochondria and affect mtDNA replication or repair. These other polymerases could be additional targets for ant-AIDS antiviral nucleotide analogs. Since 1970, the DNA polymerase gammaPolG) has been known to be the DNA polymerase responsible for replication and repair of mitochondrial DNA, and until recently it was generally accepted that this was the only polymerase present in mitochondria. However, recent data has challenged that opinion, as several polymerases are now proposed to have activity in mitochondria. To date, their exact role of these other DNA polymerases is unclear and the amount of evidence supporting their role in mitochondria varies greatly. To gain an appreciation of these newly proposed DNA polymerases in the mitochondria, we review the evidence and standards needed to establish the role of a polymerase in the mitochondria. Employing PolG as an example, we established a list of criteria necessary to verify the existence and function of new mitochondrial proteins. We then apply this criteria towards several other putative mitochondrial polymerases. While there is still a lot left to be done in this exciting new direction, it is clear that PolG is not acting alone in mitochondria, opening new doors for potential replication and repair mechanisms. DNA polymerase theta (POLQ) is a unique A-family polymerase that we discovered in 1999 and is essential for alternative end-joining (alt-EJ) of double-strand breaks (DSBs) and performs translesion synthesis. Recently, POLQ was implicated in mtDNA maintenance in a screen for genes involved in rescuing mtDNA damage upon mitochondrial derived oxidative stress. POLQ was further shown to be mitochondrial by identification in enriched mitochondrial fractions and protection from degradation by proteases in these fractions, as well as immunofluorescence co-localization with MitoTracker Red and punctate with nucleoids. POLQ, like pol gamma, is sensitive to dideoxynucleosides and similar antiviral drugs such as D4T. The in vivo consequences of POLQ inhibition by these analogs is unknown. POLQ is also highly expressed in cancer cells, confers resistance to ionizing radiation and chemotherapy agents, and promotes the survival of homologous recombination (HR) deficient cells, and thus represents a promising new cancer drug target. As a result, identifying substrates that are selective for this enzyme is a priority. In collaboration with Richard Pomerantz (Temple University) we demonstrate that POLQ efficiently and selectively incorporates into DNA large benzo-expanded nucleotide analogs (dxAMP, dxGMP, dxTMP, dxAMP) which exhibit canonical base-pairing and enhanced base stacking. In contrast, functionally related Y-family translesion polymerases exhibit a severely reduced ability to incorporate dxNMPs, and all other human polymerases tested from the X, B and A families fail to incorporate them under the same conditions as POLQ. We further find that POLQ is inhibited after multiple dxGMP incorporation events, and that POLQ efficiency for dxGMP incorporation approaches that of native dGMP. These data demonstrate a unique function for POLQ in incorporating synthetic large-sized nucleotides and suggest the future possibility of the use of dxG nucleoside or related prodrug analogs as selective inhibitors of POLQ activity. In 1998, in collaboration with the Wilson group, we discovered that human PolG like pol beta has dRP lyase activity. This activity is thought to be critical to mitochondrial BER. We subsequently identified a similar dRP lyase activity in POLQ and delineated the active site Lys to a 23 kDa fragment containing the polymerase domain. In 2015, Dr. Caglayan in the Wilson group discovered that pol beta has a 5deadenylation activity that can complement aprataxin deficiency. This prompted us to ask whether PolG could also perform this activity, and Dr. Caglayan found that PolG harbored a weak deadenylation activity. By analysis of highly purified mitochondrial extracts we determined that the in vivo deadenylation activity of PolG was insufficient to complement aprataxin deficiency. However, one of the problems we encountered during the preparation of highly purified mitochondrial extracts was contaminating pol beta activity and only upon using pol beta -/- DT40 cells or pol beta -/- MEF cells, were we able to ascertain that PolG deadenylation activity was insufficient in vivo. This idea prompted us (the Wilson and Copeland groups) to question the evidence about pol beta in the mitochondria. Highly purified mitochondrial extracts were prepared via sucrose gradients followed by percoll gradients and then treated with proteases or DNase I before lysis of mitochondria. These preparations exhibited base excision repair activity that was dependent on pol beta. Mitochondria from pol beta-deficient mouse fibroblasts had compromised DNA repair and showed elevated levels of superoxide radicals after hydrogen peroxide treatment. Mitochondria in pol beta-deficient fibroblasts displayed altered morphology by electron microscopy. These results suggest mammalian mitochondria contain a base lesion repair system mediated by pol beta. The finding that pol beta is present in the mitochondria has implications in our understanding of NRTI toxicity in mitochondria. In 1992, Copeland found that pol beta, like PolG, is highly sensitive to dideoxynucleotides but not AZT. Thus, pol beta, like PolG, should now be considered as a potential target of NRTI induced mitochondrial toxicity.